Clinical Trials of Non-Coding RNAs as Diagnostic and Therapeutic Biomarkers for Central Nervous System Injuries

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INTRODUCTION
Central nervous system (CNS) injuries, including brain injury and spinal cord injury (SCI), often cause irreversible neurological damage and high medical care expenditure.In 2016, the worldwide agestandardized incidence rates of traumatic brain injury (TBI) and SCI were 369 and 13 per 100 000, respectively, with an estimated 27.08 million new cases of TBI and 0.93 million new cases of SCI [1].While a diagnosis of severe TBI is generally uncomplicated, mild TBI (mTBI), especially mild closed head injury, is more challenging to detect.Noncoding RNAs (ncRNAs) have previously been considered as potential noninvasive diagnostic biomarkers or drug targets for several human diseases [2,3].In TBI and SCI, a series of ncRNAs, such as microRNAs (miRNAs), long-coding RNAs (lncRNAs), small nucleolar RNA (snoRNA), and wiRNA have been evaluated for their potential use as diagnostic and therapeutic biomarkers [4][5][6][7][8][9][10][11].In this review, we summarize current advances in the application of ncRNAs as diagnostic and prognostic markers of CNS injuries based on data from clinical trials.The potential roles of ncRNAs in axon regeneration after CNS injuries are introduced.Additionally, the challenges and current directions of clinical studies are also discussed.

CLINICAL TRIALS OF ncRNAs AS BIOMARKERS IN THE DIAGNOSIS AND PROGNOSIS OF CNS IN-JURIES
The ClinicalTrials.govdatabase (https://clinicaltrials.gov/) currently lists a total of 49 clinical trials evaluating the potential role of ncRNAs as diagnostic and therapeutic biomarkers for CNS injuries (Table 1).Among these trials, 20 focus on stroke, five on subarachnoid hemorrhage, nine on TBI, five on Parkinson's disease (PD), five on epilepsy, three on amyotrophic lateral sclerosis (ALS), and two on SCI.As of July 20, 2022, 21 of the studies are still recruiting participants, 13 have been completed, and 3 were terminated.The estimated sample sizes used in the clinical trials exhibited large variations, ranging from 5 to 1,620.Only 12 studies were clinical phase trials, the majority (66.7%) being clinical phase II.
Delayed cerebral infarction (DCI) is a common complication of subarachnoid hemorrhage (SAH) with high morbidity and mortality.Multiple studies have been conducted to determine the extent to which miRNA expression can serve as a diagnostic tool for SAH.Results from two case-control studies (NCT03344744 and NCT01791257) showed that miR-21, miR-221, miR-4532, miR-4463, miR-1290, and miR-4793 may differentiate SAH patients with DCI from those without DCI [15,16].When combining miR-4532, miR-4463, miR-1290, and miR-4793, the AUC reached 0.82 (Table 2) [16].However, the study had a relatively small sample size (20 SAH patients with DCI and 20 SAH patients without DCI), and the results should be confirmed in larger replication studies.
Given that the diagnosis of mild TBI (mTBI) remains a critical challenge due to subtle signs and syndromes and the absence of reliable and objective biomarkers, several studies have been conducted to assess the value of ncRNAs in the diagnosis and outcome prediction of mTBI.For example, a prospective observational study (NCT02639923) found that plasma levels of miR-92a and miR-16 were significantly increased in mTBI patients within the first 24 hours postinjury, with AUC values of 0.78 and 0.82 for the two miR-NAs, respectively [10].In addition to plasma-induced ncRNAs, salivary ncRNAs may alternatively be used to discriminate mTBI from healthy controls.In 2016, a multicenter clinical trial (NCT02901821) was carried out to assess the ability of ncRNAs to predict symptom duration and character following mTBI.The study recruited 538 individuals (251 mTBI patients and 287 controls) who were divided into testing and training groups.Random forest was performed to create an mTBI-predictive algorithm based on datasets of single ncRNA and ncRNA ratios.The age of participants and seven ncRNA ratios were included in the ncRNA model, and the AUC for differentiating mTBI from controls was 0.86 in the training set and 0.82 in the testing set (95% confidence interval (CI): 0.82-0.90).Using an expanded model combining clinical variables (age of participants, symptom severity, symptom burden) and the expression of miR-4510, miR-27a-5p, miR-1246, and wiRNA_2048, the AUC for differentiating mTBI from controls was 0.93 (95% CI: 0.89-0.97)[17].Furthermore, machine learning techniques were used to predict the status of persistent post-concussion symptoms.The prognostic algorithm included 16 ncRNAs and the age of participants, and reached an AUC of 0.83 (95% CI: 0.81-0.85).When identifying symptom recovery at 21 days postinjury, the best ncRNA model consisted of miR-12136, miR-200a-5p, miR-203a-5p, miR-423-5p, wiRNA_3506, wiR-NA_9363, wiRNA_9447, wiRNA_10135, RNU1-1, RNVU1-17, SNORD18B, and age of participants (Table 2) [18].

CONCLUSION
Over the past two decades, the tissue-specific and differential expression features of ncRNAs have been studied extensively, and ncRNAs have emerged as highly potential targets for the diagnosis and treatment of CNS injuries.For example, the combination of miR-4510, miR-27a-5p, miR-1246, and wiRNA_2048 expression as a disease biomarker was found to have more than 90% accuracy in detecting mTBI [17], while the combination of linc-DHFRL1-4, SNHG15, and linc-FAM98A-3 expression had more than 80% accuracy in detecting stroke [12].Despite these promising results, however, there are still some challenges that must be overcome before ncRNAs may be effectively implemented in clinical practice: (1) hundreds or thousands of ncRNAs have been identified to date, and the ncRNAs evaluated in different clinical trials are of diverse types.But there is still no widely accepted set of ncRNAs that are considered to be most suitable for clinical application; (2) most clinical trials to date have evaluated the diagnostic and prognostic value of ncRNAs in CNS injuries, but no study has yet assessed the therapeutic potential of ncRNAs as drug targets; (3) intracerebral drug delivery presents several challenges which further hamper clinical implementation.Exosomal ncRNAs carried by novel biomedical materials or intranasal delivery may provide new opportunities for the treatment of CNS injuries.
Until now, more than half of the clinical trials registered in the ClinicalTrials.govdatabase are ongoing.After these studies have been completed, more evidence will be provided, which might open new avenues for the diagnosis and prognosis of CNS injuries.Moreover, the exploration of novel CNS therapeutic drugs targeting ncRNAs will be of great importance in future clinical trials.

AUTHORS' CONTRIBUTIONS
WH drafted the manuscript.WQ, XX, and LX helped to revise the manuscript.GL designed the project and revised the manuscript.

Fig. ( 1 ).
Fig. (1).The potential roles and interactive networks of ncRNAs in neurite outgrowth and axon regeneration following CNS injury.(A higher resolution/colour version of this figure is available in the electronic copy of the article).